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383
JPP 2005, 57: 383–391
ß 2005 The Authors
Received June 15, 2004
Accepted November 29, 2004
DOI 10.1211/0022357055605
ISSN 0022-3573
Department of Physiology,
School of Medicine, University of
Santiago de Compostela, 15782
Santiago de Compostela, Spain
E. Vigo, A. Cepeda,
R. Perez-Fernandez
Laboratorio de Investigacio´n4,
Area de Docencia e
Investigacio´ n, Hospital Clı´nico
Universitario, 15782 Santiago
de Compostela, Spain
O. Gualillo
Correspondence: R. Perez-
Fernandez, Departamento de
Fisiologı´a, Facultad de Medicina,
Universidad de Santiago de
Compostela, 15782
Santiago de Compostela,
Spain. E-mail: fsropefe@usc.es
Funding: This work was
supported in part by a I þ D
research grant (2000/CE535)
from Bioserum Laboratorios,
S.L. Oreste Gualillo is a recipient
of a research contract 00/3051
from the Instituto de Salud
Carlos III, Ministerio de Sanidad
y Consumo, Spain.
In-vitro anti-inflammatory activity of Pinus sylvestris
and Plantago lanceolata extracts: effect on inducible
NOS, COX-1, COX-2 and their products in J774A.1
murine macrophages
E. Vigo, A. Cepeda, O. Gualillo and R. Perez-Fernandez
Abstract
Extracts of the plant species Pinus sylvestris L. and Plantago lanceolata L. have been used in tradi-
tional medicine for the treatment of certain respiratory diseases, but little is known about their
precise effects and mechanisms o f action. In this study, we investigated the effect of these plant
extracts on the production of nitric oxide (NO) and prostaglandin E
2
, NO synthase (NOS) type II,
cyclooxygenase-1 (COX-1) and COX-2 mRNA expression in the murine macrophage cell line J774A.1.
We found that Pinus sylvestris and Plantago lanceolata extracts inhibited NO production in a
concentration-dependent manner in this cell line, without obvious cytotoxic effects as tested by
MTT assay. The Plantago lanceolata extract at all doses u sed, and the Pinus sylvestris extract at high
doses, showed significant scavenging of NO radicals released by the NO donor PAPA-NONOate. Our
data also show that pre-treatment with these extracts significantly inhibits inducible NOS (iNOS)
mRNA production in this cell line, without affecting COX-1 mRNA expression. COX-2 mRNA levels
and PGE
2
levels induced by lipopolysaccharide/interferon- were not modified upon pre-treatment
with the extracts. Thus, our results suggest that the anti-inflammatory properties of Pinus sylvestris
and Plantago lanceolata extracts may reflect decreased NO production, possibly due to inhibitory
effects on iNOS gene expression or to NO-scavenging activity.
Introduction
Inflammation is a complex process characterized by the contribution of various
mediators, including prostaglandins and nitric oxide (NO) (Higgs et al 1984; Nathan
1997). Cyclooxygenase (COX) is one of the main enzymes involved in the metabolism
of arachidonic acid, catalysing the synthesis of prostaglandins and thromboxane
(Smith et al 1991). COX exists in two isoforms: COX-1 is a ubiquitously and consti-
tutively expressed isoform that is postulated to have housekeeping functions; COX-2 is
an inducible isoform that has been implicated in inflammatory responses and the
regulation of cell growth and differentiation (Smith et al 1996). Specifically, COX-2
is thought to be the primary generator of the prostanoids that contribute to inflam-
mation, acting in both the inflammation initiation and resolution phases (Gilroy et al
1999). However, several studies have shown that prostanoids formed via COX-1 are
also involved in inflammation processes (Langenbach et al 1995; Wallace et al 1998).
The pivotal role of nitric oxide (NO) as a messenger and effector molecule has been
demonstrated in a variety of tissues (Palmer et al 1988; Lowenstein et al 1996). NO has
been identified as a neurotransmitter in the central nervous system and a potent
physiological vasorelaxant that regulates blood pressure by modulating muscular
tone (Hibbs et al 1987; Moncada et al 1991). NO is also an important molecule in
inflammation and sepsis (Wheeler & Bernard 1999). Exposure to bacterial surface
molecules, such as lipopolysaccharide (LPS) and lipoteicholic acid (LTA), stimulates
cellular inflammatory responses and induces release of pro-inflammatory factors,
including NO, prostaglandin E
2
(PGE
2
), cytokines, tumour necrosis factor- and
eicosanoid mediators. At least three types of nitric oxide synthase (NOS) isoform
have been identified in cells. Both the endothelial nitric
oxide synthase (eNOS) and neural nitric oxide synthase
(nNOS) isoforms are constitutive (cNOS), in that NO
produced by cNOS contributes to maintaining the normal
active state of vasodilatation through a Ca
2þ
/calmodulin-
dependent pathway, and acts as a neurotransmitter in
neuron signal transmission. NOS in macrophages and
hepatocytes is inducible (the iNOS isoform), and its acti-
vation is Ca
2þ
/calmodulin-independent. Exposure to
endogenous and exogenous stimulators induces iNOS in
various cells, such as macrophages, smooth muscle cells
and hepatocytes, triggering various detrimental cellular
responses and potentially causing disease, including
inflammation, sepsis and stroke (Marletta et al 1988;
Nathan 1992; Marletta 1993; Duval et al 1996). NO pro-
duction induced by iNOS may thus reflect the degree of
inflammation, and provides a useful way of assessing the
effect of drugs on the inflammatory process. Conversely,
inhibition of NO accumulation induced by inflammatory
stimuli could be a useful strategy for treatment of inflam-
matory diseases (Hobbs et al 1999).
Pinus sylvestris L. and Plantago lanceolata L. extracts
have been used in traditional medicine as anti-inflamma-
tory treatments in bronchitis, asthma and other respira-
tory diseases (Matev et al 1982; Peris et al 1995;
Blumenthal 1998). It has recently been demonstrated
that bioflavonoids extracted from the bark of Pinus mar-
itima inhibit the expression of the pro-inflammatory cyto-
kine interleukin-1 (IL-1) by regulating redox-sensitive
transcription factors, namely nuclear factor-B (NF-B)
and activating protein-1 (AP-1), in LPS-stimulated RAW
264.7 murine monocyte macrophages (Cho et al 2000).
Pinus maritima bark extract is also a powerful scavenger
of reactive oxygen and nitrogen species (Virgili et al 1998).
It has additionally been demonstrated that a hexane
extract of Plantago major (ursolic acid) inhibits COX-2;
this would thus explain the anti-inflammatory effects of
Plantago major extracts (Ringbom et al 1998) and is in
accordance with previous results indicating that Plantago
lanceolata (probably phenylethanoid components thereof)
inhibits arachidonic-acid-induced mouse ear oedema
(Murai et al 1995). In this study, to further characterize
the anti-inflammatory properties of Pinus sylvestris and
Plantago lanceolata extracts, we investigated their effects
at different doses on cell viability, NO production, scaven-
ging activity, iNOS mRNA expression, COX-1 and -2
mRNA expression and PGE
2
levels in the murine macro-
phage line J774A.1.
Materials and Methods
Materials
The Pinus sylvestris extract and the Plantago lanceolata
extract were obtained from Bioserum Laboratorios S.L.
(Malaga, Spain). The Pinus sylvestris extract was prepared
as follows. Leaf buds were subjected to steam distillation for
extraction of essential oils, which were set aside. The same
leaf buds were then percolated in ethanol–water (45:55) at
40
C for 10 h, then concentrated in a vacuum concentrator.
The essential oils and the percolation concentrate were
pooled to give the final extract, to a total essential oils
concentration of 1% w/w pinene equivalent and a total
flavonoids concentration of 0.3% w/w rutin equivalent.
Total essential oils were determined by gas/liquid chroma-
tography (GLC) (Shimazu model GC-14A; 30-cm SP-5
column; detection temp 200
C, injection temp. 200
C; col-
umn temp. first 50
C, then linear gradient of 5
Cmin
1
to
110
C; nitrogen carrier 2 kg cm
2
; injection volume 3 mL;
FID detection). Total flavonoids were determined by
reverse-phase high-performance liquid chromatography
(HPLC) (Hewlett Packard model HP1050; 150 3.20 mm
Prodigy 5 mm ODS(3) 100A column; mobile phase metha-
nol–phosphate buffer 5 m
M, pH 7 (40:60, v/v); injection
volume 2 mL; flow rate 1 mL min
1
; detection at 365 nm).
Finally, the extract was dried by atomization.
The Plantago lanceolata extract was prepared as fol-
lows. Whole plants (leaves, flowers and roots) were per-
colated in ethanol–water (45:55) at 40
C for 10 h, then
concentrated in a vacuum concentrator to give the final
extract. Tannin concentration in the extract, determined
spectrophotometrically at 715 nm in a Beckman DU-40
apparatus, was standardized to 2% w/w. Again, the
extract was dried by atomization.
For experiments, the powdered extracts of Pinus syl-
vestris and Plantago lanceolata were dissolved in ethanol–
water (1:24). Both extracts were used at a stock concen-
tration of 1.68 gmL
1
. For control experiments only the
ethanol–water vehicle was used.
The J774.1 cell line was obtained from the European
Collection of Cell Cultures (Salisbury, UK). Dulbecco’s
Modified Eagle’s Medium (DMEM) was obtained from
Gibco-BRL (NY). LPS (E. coli 0111:B4), interferon-
(IFN-), dexamethasone, indometacin and PAPA-
NONOate were obtained from Sigma (St Louis, MO).
3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT, 5 mg mL
1
) was purchased from Roche
Diagnostics Corporation (Indianapolis, USA). All
reagents were of analytical grade. Absorbance was mea-
sured using a microplate reader (BioRad 550; Bio-Rad
Laboratories, Hercules, CA).
Cell culture and stimulation of macrophages
with LPS and IFN-g
The J774A.1 cell line was maintained in DMEM contain-
ing 10% fetal bovine serum (FBS), 2 m
ML-glutamine,
100 U mL
1
penicillin, 100 gmL
1
streptomycin and
130 gmL
1
sodium pyruvate, at 37
C under 5% CO
2
humidified air. Cells were harvested by gentle scraping
and passaged every 3–6 days by 1:6 dilution. For stimula-
tion with LPS and IFN-, cells were seeded into 24-well
plates at a density of 1 10
5
cells/well and allowed to
adhere for 12 h at 37
C under 5% CO
2
atmosphere.
Culture medium was then replaced with fresh medium
without FBS for 5 h, then replaced again with fresh med-
ium containing 10% FBS, 1 gmL
1
LPS and 15 ng mL
1
IFN- in phosphate-buffered saline (PBS). Doses of LPS
384 E. Vigo et al
and IFN- were chosen on the basis of preliminary find-
ings indicating that they give optimal induction of induci-
ble nitric oxide synthesis in J774A.1 cells. To evaluate the
effects of the extracts, cells were first incubated for 5 h
with the Pinus sylvestris or Plantago lanceolata extract
(8.5, 16.8, 50.4 or 84 gmL
1
), then for 24 h with LPS
plus IFN- as above. As reference controls, assays were
also performed with the anti-inflammatory steroid dexa-
methasone (0.01, 0.1 or 1
M) and the non-steroidal anti-
inflammatory drug indometacin (0.01, 0.1 or 0.25 m
M).
Measurement of nitrite production
As an indicator of NO production, we determined the
nitrite concentration in the culture medium by the Griess
reaction (Dirsch et al 1998). One hundred microlitres of
each culture supernatant, assayed in triplicate, was reacted
with an equal volume of Griess reagent (1% sulfanilamide
and 0.1% naphthylethylenediamine HCl in 2.5% phospho-
ric acid) at room temperature for 10 min. Absorbance was
then measured at 540 nm, and nitrite concentration deter-
mined using sodium nitrite as standard.
MTT assay for cell viability
Cell viability was assessed by the mitochondrial-respira-
tion-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) reduction method. Cells
(1 10
4
cells/well) in 96-well plates were incubated with
increasing doses of test compound (8.5, 16.8, 50.4 or
84 gmL
1
of Pinus sylvestris or Plantago lanceolata
extract; 0.01, 0.1 or 1
M of dexamethasone; or 0.01, 0.1
or 0.25 m
M of indometacin) at 37
Cin5%CO
2
for 24 h.
After treatment, 10 L of MTT solution was added to
each well. After incubation for 4 h at 37
C, the formazan
crystals in viable cells were solubilized with 100 L of lysis
buffer (10% sodium dodecyl sulfate in 0.01
M HCl) for
12 h. The absorbance of each well was then read at
540 nm. The optical density of formazan formed in control
(untreated) cells was taken as 100% viability.
Assay of scavenging of NO radicals released
by PAPA-NONOate
The plant extracts (8.5, 16.8, 50.4 or 84 gmL
1
of Pinus
sylvestris or Plantago lanceolata extract) were dissolved in
PBS to a total volume of 250 L and incubated with
250 L of PAPA-NONOate dissolved in PBS (pH 7.68,
20
M)at37
C for 3 h. After incubation, the concentration
of nitrite was measured by the Griess method as described
above.
Assay of iNOS, COX-1 and COX-2 mRNA expression:
isolation and reverse transcription-polymerase
chain reaction (RT-PCR) amplification
The J774A.1 macrophage cell line (1 10
6
cells) was
grown in a 90-mm petri dish as described above. The
cells were treated with the plant extracts (84 gmL
1
for
Pinus sylvestris L. and Plantago lanceolata L.) and 5 h
later LPS plus IFN- (1 gmL
1
and 15 ng mL
1
, respec-
tively) was added. Total RNA was isolated with TRIzol
reagent (Invitrogen, Life Technologies, UK) as previously
described (Gil-Puig et al 2002). RNA concentration and
purity were determined by spectrophotometry. cDNA
synthesis and PCR amplification of GADPH and iNOS
were performed as previously described (Vigo et al 2004).
For PCR amplification of COX-1 and COX-2, samples
were denatured at 94
C for 1 min, annealed at 55
C for
1 min and extended at 72
C for 1 min, for 28 cycles, with
an extension step of 10 min at 72
C in the last cycle.
To determine the relative amounts of iNOS, COX-1
and COX-2 mRNAs in each sample, iNOS, COX-1 and
COX-2 mRNA amounts were standardized with respect
to GADPH mRNA amounts. Specifically, PCR products
were separated on 2% agarose gel, stained with ethidium
bromide, visualized with UV light and quantified using
the Gel Doc 1000 Documentation System (Bio-Rad
Laboratories, CA).
Primer sequences for PCR amplification of iNOS were
as follows: primer A (5
0
-GCCTCCCTCTGGAAAGA-3
0
)
was a 17-mer corresponding to residues 1213–1230 of the
rat coding sequence, and primer B (5
0
-TCCATGCAGA
CAACCTT-3
0
) was an antisense 17-mer corresponding
to residues 1696–1712 of the coding sequence. The PCR
product obtained was 499 bp in length. Primer sequences
for COX-1 were as follows: the forward primer 5
0
-
CGGTGCGGTCCAACCTTATCC-3
0
, corresponding to
residues 411–431 of the rat coding sequence, and the reverse
primer 5
0
-CCGCAGGTGATACTGTCGTT-3
0
, corres-
ponding to residues 774–793 of the coding sequence. The
PCR product obtained was 382 bp in length. Primer
sequences for COX-2 were as follows: the forward primer
5
0
-GGGAAGCCTTCTCCAACC-3
0
, corresponding to resi-
dues 498–515 of the rat coding sequence, and the reverse
primer 5
0
-GAACCCAGGTCCTCGCTT-3
0
, corresponding
to residues 725–742 of the coding sequence. The PCR pro-
duct obtained was 245 bp in length. Primer sequences for rat
GADPH were as follows: the forward primer 5
0
-TGAT
GACATCAAG AAGGTGGTGAAG-3
0
, corresponding to
residues 758–782 of the rat coding sequence, and the reverse
primer 5
0
-TCCTTGGAGGCCATGTAGGCCAT-3
0
corres-
ponding to residues 974–997 of the coding sequence. The
PCR product obtained was 309 bp in length.
PGE
2
immunoassay
The culture medium of control and treated cells was col-
lected, centrifuged and stored at 70
Cuntiltested.The
level of PGE
2
released into culture medium was quantified
using a specific enzyme immunoassay (EIA) according to the
manufacturer’s instructions (Amersham Biosciences, UK).
Statistical analysis
Each experiment was performed at least three times with
at least 3 replicates: the minimum total number of repli-
cates was 12 (3 experiments 4 replicates). All values
are expressed as means s.d. Means were compared by
Anti-inflammatory effect of Pinus sylvestris and Plantago lanceolata 385
one-way analysis of variance with the Dunnet’s multiple
comparison test for post-hoc comparisons. Statistical sig-
nificance is taken to be indicated by P < 0.05.
Results
Effect of dexamethasone, indometacin, Pinus
sylvestris extract and Plantago lanceolata
extract on NO production by LPS/IFN-g -stimulated
J774.1 cells
Incubation of the cells with LPS plus IFN- resulted in an
increase in NO concentration in culture medium ranging
from 4.7 to 5.8
M after 24 h incubation, versus no detect-
able NO in non-treated cultures. These values (controls)
were considered as the maximal increase in NO, for calcu-
lation of the reduction in NO release by LPS/IFN--sti-
mulated cells following pre-treatment with Pinus sylvestris
extract, Plantago lanceolata extract, dexamethasone or
indometacin (Table 1).
Pre-treatment with dexamethasone at 0.1
M or higher
induced a significant reduction (P < 0.001) in NO produc-
tion. From this concentration up, the observed effect was
dose dependent (Table 1). Pre-treatment with indometacin
induced a significant reduction (P < 0.001) in NO produc-
tion at concentrations of 0.1 m
M or higher (Table 1).
Pre-treatment with the Pinus sylvestris or Plantago lan-
ceolata extracts (8.5–84 gmL
1
) in all cases significantly
reduced NO production, and the effect of both extracts
was dose dependent (Table 1).
Effect of dexamethasone, indometacin, Pinus
sylvestris extract and Plantago lanceolata
extract on cell viability
To rule out possible cytotoxic effects of the Pinus sylvestris
and Plantago lanceolata extracts in the absence of
LPS þ IFN-, we used an MTT assay. Both the Pinus
sylvestris and the Plantago lanceolata extracts significantly
reduced cell viability at the low and intermediate concen-
trations tested (8.5, 16.8 and 50.4 gmL
1
) (Table 1);
however, no significant reduction was caused by the high-
est concentration tested (84 gmL
1
). Dexamethasone
significantly reduced cell viability at the higher concentra-
tions tested (0.1 and 1
M), while indometacin had no
significant effect, except at the highest concentration
tested (0.25 m
M).
Scavenging of NO by the Pinus sylvestris and
Plantago lanceolata extracts
As noted, pre-treatment of LPS/IFN--stimulated cells
with the Pinus sylvestris or Plantago lanceolata extracts
led to a significant reduction in nitrite levels in the med-
ium. To assess whether this reduction was due to NO
scavenging, we performed assays of scavenging activity
using PAPA-NONOate for NO generation. The
Plantago lanceolata extract, at all doses tested, had signi-
ficant NO-scavenging activity, whereas the Pinus sylvestris
extract showed NO-scavenging activity only at the highest
concentration tested (84 gmL
1
) (Table 1).
Effect of the Pinus sylvestris and Plantago
lanceolata extracts and dexamethasone
on iNOS mRNA levels
To investigate whether Pinus sylvestris and Plantago lan-
ceolata extracts affect iNOS gene expression, RT-PCR
was carried out using specific primers for the iNOS and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
genes. PCR amplification of cDNA from J774A.1 cells
yielded a 499-bp product corresponding to mouse iNOS
and a 309-bp product corresponding to mouse GAPDH
(Figure 1C). Non-stimulated J774A.1 cells showed barely
detectable iNOS mRNA levels (0.15 0.08, relative
iNOS/GAPDH value; Figure 1C, lane 3). Cells incubated
for 24 h with LPS and IFN- showed markedly and sig-
nificantly higher levels (0.80 0.13, P < 0.001 with
respect to non-stimulated cells). Pre-treatment of cells
with the Pinus sylvestris or Plantago lanceolata extract
(84 gmL
1
) significantly reduced (P < 0.05) iNOS
mRNA levels with respect to the cells treated with LPS
and IFN- only (0.62 0.07 and 0.7 0.03, respectively;
Figure 1C, lanes 5 and 6, and Figure 1D). Dexamethasone
treatment likewise significantly reduced (P < 0.05) iNOS
Table 1 Effects of Pinus sylvestris extract, Plantago lanceolata
extract, dexamethasone and indometacin on NO production, cell
viability and (plant extracts only) NO-scavenging activity
Nitrite
(m
M)
Cell viability
(absorbance)
NO scavenging
(mM)
Pinus sylvestris L.
Control 5.20 0.38 0.430 0.777 0.0120 0.0077
8.5 gmL
1
2.30 0.52*** 0.320 0.027*** 0.0100 0.0068
16 gmL
1
2.20 0.68*** 0.330 0.033*** 0.0090 0.0061
50.4 gmL
1
1.60 0.41*** 0.360 0.041* 0.0090 0.0061
84 gmL
1
1.10 0.64*** 0.380 0.041 0.0080 0.0051*
Plantago lanceolata L.
Control 4.70 1.21 0.420 0.064 0.0100 0.0066
8.5 gmL
1
2.70 0.70*** 0.310 0.059*** 0.0100 0.0062*
16 gmL
1
2.60 0.83*** 0.360 0.058*** 0.0090 0.0061**
50.4 gmL
1
2.10 0.64*** 0.380 0.065* 0.0090 0.0058**
84 gmL
1
2.10 0.90*** 0.390 0.064 0.0080 0.0050**
Dexamethasone
Control 5.80 0.43 0.52 0.24
0.01
M 5.20 0.48 0.50 0.22
0.1
M 4.70 1.10*** 0.49 0.19*
1
M 2.90 1.58*** 0.47 0.19***
Indometacin
Control 5.30 0.56 0.65 0.12
0.01 m
M 4.10 0.18 0.600 0.099
0.1 m
M 3.10 0.64*** 0.620 0.046
0.25 m
M 1.90 0.65*** 0.580 0.082*
***P < 0.001, **P < 0.01 and *P < 0.05 with respect to control.
386 E. Vigo et al
mRNA levels with respect to cells treated with LPS and
IFN- only (Figure 1A, B).
Effect of the Pinus sylvestris and Plantago
lanceolata extracts on COX-1 and COX-2
mRNA levels
To investigate whether Pinus sylvestris and Plantago lanceo-
lata extracts affect COX-1 or COX-2 gene expression, RT-
PCR was carried out using specific primers for the COX-1,
COX-2 and GAPDH genes. PCR amplification of cDNA
from J774A.1 cells yielded a 382-bp product corresponding
to mouse COX-1, a 245-bp product corresponding to
mouse COX-2 and a 309-bp product corresponding
to mouse GAPDH (Figure 2A, C). Non-stimulated
J774A.1 cells showed detectable COX-1 mRNA levels
(0.55 0.14, relative COX-1/GAPDH value; Figure 2A,
lane 1). Pre-treatment of cells with the Pinus sylvestris or
Plantago lanceolata extract (84 gmL
1
) reduced COX-1
mRNA levels with respect to the control cells (0.4 0.11
and 0.41 0.08, respectively; Figure 1A, lanes 2 and 3, and
Figure 1B), though these reductions were not statistically
significant. Non-stimulated J774A.1 cells showed detectable
COX-2 mRNA levels (0.48 0.27, relative COX-2/
GADPH value). Cells incubated for 24 h with LPS and
IFN- showed a significant increase in COX-2 levels
B
0
0.2
0.4
0.6
0.8
∗
∗
Relative iNOS/GAPDH values
Control LPS +
IFN
-γ
LPS +
IFN
-γ
0.01 0.1 1
DXM (µ M)
∗
iNOS
GAPDH
A
1
CD
Relative iNOS/GAPDH values
Control Pinus s. Plantago l.
84 µ g mL
–1
0.1
0.3
0.5
0.7
0.9
∗
∗
iNOS
GAPDH
723 45 6
12 3 4 5 6
Figure 1 RT-PCR analysis of iNOS mRNA expression and GAPDH mRNA expression in activated J774A.1 macrophages following pre-
treatment with Pinus sylvestris or Plantago lanceolata extract. A. Lane 1, molecular weight markers (1000-bp DNA ladder); lane 2, negative
control of PCR; lane 3, non-stimulated cells; lane 4, LPS/IFN--stimulated cells; lanes 5–7, LPS/IFN--stimulated cells pre-treated with 0.01,
0.1 or 1
M of dexamethasone, respectively. B. Relative iNOS mRNA levels (iNOS/GADPH) in non-treated cells, LPS/IFN--stimulated cells
and dexamethasone (DXM)-pre-treated LPS/IFN--stimulated cells. C. Lanes 1–4, as for A; lane 5, LPS/IFN--stimulated cells pre-treated
with 84 gmL
1
of Pinus sylvestris extract; lane 6, LPS/IFN--stimulated cells pre-treated with 84 gmL
1
of Plantago lanceolata extract.
D. Relative iNOS mRNA levels (iNOS/GADPH) in non-treated cells, LPS/IFN--stimulated cells and Pinus sylvestris-orPlantago lanceolata-
pretreated LPS/IFN--stimulated cells (*P < 0.05 with respect to cells treated with LPS and IFN- only).
Anti-inflammatory effect of Pinus sylvestris and Plantago lanceolata 387
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
1
2
3
4
5
6
7
8
9
A
B
D
C
E
Relative COX-1/GAPDH values
Relative COX-2/GAPDH values
PGE
2
(pg/well)
COX-1
COX-2
GAPDH
GAPDH
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
Control Pinus s. Plantago l.
∗
∗
∗
∗
∗
∗
Control LPS +
IFN-
γ
Pinus s. Plantago l.
84 µ g mL
–1
84 µ g mL
–1
Control LPS +
IFN-
γ
Pinus s. Plantago l.
84 µ g mL
–1
Figure 2 RT-PCR analysis of COX-1 and COX-2 mRNA levels and GAPDH mRNA levels in activated (COX-2) or non-activated (COX-1)
J774A.1 macrophages following pre-treatment with Pinus sylvestris or Plantago lanceolata extract. A. COX-1 RT-PCR: Lane 1, non-
stimulated cells; lane 2, cells treated with 84 gmL
1
of Pinus sylvestris extract; lane 3, cells treated with 84 gmL
1
of Plantago lanceolata
extract; lane 4, negative control of PCR; lane 5, molecular weight markers (1000-bp DNA ladder). GAPDH RT-PCR: lanes 6, 7, 8 and 9, as for
4, 1, 2 and 3, respectively. B. Relative COX-1 mRNA levels (COX-1/GADPH) in non-treated cells (control) and cells pre-treated with Pinus
sylvestris or Plantago lanceolata extract. C. COX-2 RT-PCR: lane 1, non-stimulated cells; lane 2, LPS/IFN--stimulated cells; lanes 3 and 4,
LPS/IFN--stimulated cells pre-treated with 84 gmL
1
of Pinus sylvestris or Plantago lanceolata extract. GAPDH RT-PCR: lanes 6, 7, 8 and
9, as for lanes 1, 2, 3 and 4. D. Relative COX-2 mRNA levels (COX-2/GADPH) in non-treated cells (control), in LPS/IFN--stimulated cells
and in LPS/IFN--stimulated cells pre-treated with Pinus sylvestris or Plantago lanceolata extract. E. Effects of the Pinus sylvestris or Plantago
lanceolata extract on LPS/IFN--induced PGE
2
in J774A.1 macrophages (*P < 0.05 with respect to control cells).
388 E. Vigo et al
(0.87 0.3, P < 0.05, with respect to non-stimulated cells).
Pre-treatment of LPS/IFN--stimulated cells with the Pinus
sylvestris or Plantago lanceolata extract (84 gmL
1
)sig-
nificantly increased (P < 0.05) COX-2 mRNA levels with
respect to the control cells (0.94 0.28 and 0.94 0.27,
respectively; Figure 2C, lanes 3 and 4, and Figure 2D),
but not with respect to LPS/IFN--stimulated cells.
Effect of the Pinus sylvestris and Plantago
lanceolata extracts on PGE
2
levels
Treatment of J774A.1 macrophages with LPS and IFN-
caused a significant accumulation of PGE
2
(6.82 0.72
pg/well), compared with the release by unstimulated cells
(3.45 1.57 pg/well) (Figure 2E). Pre-treatment of LPS/
IFN--stimulated cells with the Pinus sylvestris or Plantago
lanceolata extract (84 gmL
1
) did not significantly affect
PGE
2
production with respect to LPS/IFN--stimulated
cells (6.2 1.65 pg/well and 6.5 0.86 pg/well, respectively).
Discussion
In this work, it is demonstrated that the enhanced produc-
tionofNOinducedbyLPSandinterferon- in a murine
macrophage cell line, J774A.1, is significantly and dose-
dependently inhibited by the previous administration of
Pinus sylvestris or Plantago lanceolata extract. In MTT
assays of effects on cell viability, we rather surprisingly
found that both the Pinus sylvestris and Plantago lanceolata
extracts reduced cell viability at low and intermediate
concentrations but not at higher concentrations; this was
confirmed by replicate assays. Regardless of possible
explanations for these apparent dual effects, these results
indicate that the observed inhibition of NO production by
the extracts cannot be attributed – at least at high concen-
trations – to cytotoxicity. Furthermore, both extracts (at
least at high concentrations) showed scavenging of NO
radicals released by the NO generator PAPA-NONOate.
Our results also indicate that pre-treatment with Pinus
sylvestris or Plantago lanceolata extracts inhibits iNOS
mRNA expression without affecting COX-1 mRNA levels.
In addition, neither the Pinus sylvestris nor the Plantago
lanceolata extracts decreased COX-2 mRNA expression or
PGE
2
levels in LPS/IFN--stimulated macrophages.
The anti-inflammatory steroid drug dexamethasone
and the non-steroidal anti-inflammatory drug indometa-
cin, used as reference controls, significantly inhibited NO
production by LPS/IFN--stimulated cells, without
affecting (at least at the low dose tested) cell viability.
These results for dexamethasone and indometacin are in
accordance with previously published data (Di Rosa et al
1990; Ogawa et al 2000).
It is well known that some chemical constituents of
medicinal plants show biological activity affecting differ-
ent aspects of the inflammation process. Many flavonoids
and phenylethanoids are antioxidants (Rice-Evans et al
1996; Xiong et al 2000) and some of these compounds
are also known to show free-radical scavenging activity
(Kandaswami & Middleton 1994; Xiong et al 2000). In
this study, the Plantago lanceolata extract showed NO
scavenging activity at all the doses tested, whereas the
Pinus sylvestris extract only showed NO scavenging activ-
ity at the highest concentration. This could be due to the
fact that the scavenging activity of phenolic compounds
requires a large number of phenol groups per molecule
(Wang et al 1996; Xiong et al 1996). For example,
Plantago asiatica, a member of the same genus family as
Plantago lanceolata, does not show scavenging activity,
but significantly inhibits both NO production and iNOS
mRNA expression (Tezuka et al 2001). NO production by
macrophages depends on iNOS, which can be activated by
various agents, including interferons, tumour necrosis fac-
tor- (TNF-) and LPS (Moncada et al 1991). The onset
of the NO production cascade induced by LPS or cyto-
kines requires a number of steps, including the activation
of nuclear factor NF-B and subsequent iNOS mRNA
expression. Some flavonoids decrease iNOS protein levels
and activity and NO production by reducing the expres-
sion of iNOS mRNA, and this reduction may occur
through prevention of the binding of NF-B to the
iNOS promoter, thereby inhibiting the induction of
iNOS transcription (Lin & Lin 1997). Thus, we cannot
rule out the possibility that the observed inhibition of
transcription of the iNOS gene in LPS/IFN--stimulated
J774A.1 macrophages following pre-treatment with Pinus
sylvestris or Plantago lanceolata extracts may be mediated
by inhibition of, or interference with, NF-B. Similar
findings have been obtained for dexamethasone, which
decreases the activity of the iNOS promoter and reduces
the formation of cytokine-induced NF-B complexes that
bind to the NF-B site in the human iNOS promoter
(Kleinert et al 1996).
Macrophage activation by LPS leads to a functionally
diverse series of responses, including the activation of
phospholipase A
2
leading to the production of lipid meta-
bolites of arachidonic acid (such as prostaglandins). COX
is a rate-limiting enzyme in the conversion of arachidonic
acid to PGH
2
, the precursor of a large group of biologi-
cally active mediators, such as PGE
2
, prostacyclin and
thromboxane B
2
(TXB
2
). COX-1 is constitutively
expressed in many tissues and predominates, for example,
in gastric mucosa. Inhibition of COX-1, which reduces the
basal production of cytoprotective PGE
2
and PGI
2
in the
stomach, may contribute to gastric ulceration. In this
study, we did not observe a significant decrease in COX-1
mRNA levels in J774A.1 macrophages pre-treated with
Pinus sylvestris or Plantago lanceolata extracts, suggesting
that neither extract inhibits COX-1 expression. Pre-treat-
ment with Pinus sylvestris or Plantago lanceolata extracts
did not decrease the significantly increased COX-2
mRNA expression or PGE
2
levels observed in macro-
phages after LPS/IFN- challenge. It has been reported
previously (Liang et al 1999) that different flavonoids
affect iNOS and COX-2 expression in different ways:
apigenin reduced both iNOS and COX-2 expression,
whereas flavonoids, such as quercetin, reduced iNOS
expression but enhanced COX-2 expression. These results
suggest that the anti-inflammatory properties of Pinus
Anti-inflammatory effect of Pinus sylvestris and Plantago lanceolata 389
sylvestris and Plantago lanceolata extracts are related to an
inhibition of NO via reduced iNOS mRNA production or
via their NO scavenging activity, not via reduced COX-2
mRNA production. The fact that pre-treatment with these
extracts did not decrease COX-1 mRNA levels suggests
that these extracts will not cause gastric pathologies.
Conclusions
This study has demonstrated that pre-treatment with
Pinus sylvestris or Plantago lanceolata extracts inhibits
NO production and iNOS mRNA expression by LPS/
IFN--stimulated murine macrophages of the J774A.1
cell line. Pre-treatment with these extracts did not modify
COX-1 mRNA production or decrease the high levels of
COX-2 and PGE
2
induced by LPS/IFN- challenge, sug-
gesting that the observed anti-inflammatory properties of
Pinus sylvestris and Plantago lanceolata extracts may be
related to the inhibition of NO, not to reduced prosta-
glandin production.
References
Blumenthal, M. (1998) The complete German Commission E
monographs. Therapeutic guide to herbal medicines. American
Botanical Council, Austin, Texas, pp 185–187
Cho, K.-J., Yun, C.-H., Yoon, D.-Y., Cho, Y.-S., Rimbach, G.,
Packer, L., Chung, A.-S. (2000) Effect of bioflavonoids
extracted from the bark of Pinus maritima on proinflamma-
tory cytokine interleukin-1 production in lipopolysaccharide-
stimulated RAW 264.7. Toxicol. Appl. Pharmacol. 168: 64–71
Di Rosa, M., Radomski, M., Carnuccio, R., Moncada, S. (1990)
Glucocorticoids inhibit the induction of nitric oxide synthase
in macrophages. Biochem. Biophys. Res. Commun. 172:
1246–1252
Dirsch, V. D., Stuppner, H., Vollmar, A. M. (1998) The
Griess assay: suitable for a bio-guided fractionation of anti-
inflammatory plant extracts? Planta Med. 64: 423–426
Duval, D. L., Miller, D. R., Collier, J., Billings, R. E. (1996)
Characterization of hepatic nitric oxide synthase: identifica-
tion as the cytokine-inducible form primarily regulated by
oxidants. Mol. Pharmacol. 50: 277–284
Gil-Puig, C., Blanco, M., Garcı
´
a-Caballero, T., Segura, C.,
Perez-Fernandez, R. (2002) Pit-1/GHF-1 and GH expression
in the MCF-7 human breast adenocarcinoma cell line.
J. Endocrinol. 173: 161–167
Gilroy, D. W., Colville-Nash, P. R., Willis, D., Chivers, J., Paul-
Clark, M. J., Willoughby, D. A. (1999) Inducible cyclooxygen-
ase may have anti-inflammatory properties. Nat. Med. 5:
698–701
Hibbs, J. B., Taintor, R. R., Vavrin, Z. (1987) Macrophage
cytotoxicity: role of l-arginine deiminase and iminonitrogen
oxidation to nitrite. Science 235: 473–476
Higgs, G. A., Moncada, S., Vane, J. R. (1984) Eicosanoids in
inflammation. Ann. Clin. Res. 16: 287–299
Hobbs, A., Higgs, A., Moncada, S. (1999) Inhibition of nitric
oxide synthase as a potential therapeutic target. Ann. Rev.
Pharmacol. Toxicol. 39 : 191–220
Kandaswami, C., Middelton, E. (1994) Free radical scavenging
and antioxidant activity of plant flavonoids. Adv. Exp. Med.
Biol. 366: 351–376
Kleinert, H., Euchenhofer, C., Ihrig-Biedert, I., Forstermann, U.
(1996) Glucocorticoids inhibit the induction of nitric oxide
synthase II by down-regulating cytokine-induced activity of
transcription factor nuclear factor-B. Mol. Pharmacol. 49:
15–21
Langenbach, R., Morham, S. G., Tiano, H. F., Loftin, C. D.,
Ghanayem, B. I., Chulada, P. C., Mahler, J. F., Lee, C. A.,
Goulding, E. H., Kluckman, K. D., Kim, H. S., Smithies, O.
(1995) Prostaglandin synthase 1 gene disruption in mice
reduces arachidonic acid-induced inflammation and indo-
methacin-induced gastric ulceration. Cell 83: 483–492
Liang,Y.C.,Huang,Y.T.,Tsai,S.H.,Lin-Shiau,S.Y.,Chen,C.F.,
Lin, J. K. (1999) Supression of inducible cyclooxygenase and
nitric oxide synthase by apigenin and related flavonoids in
mouse macrophages. Carcinogenesis 20: 1945–1952
Lin, Y. L., Lin, J.-K. (1997) ()-Epigallocatechin-3-gallate blocks
the induction of nitric oxide synthesis by down-regulating
lipopolysaccharide-induced activity of transcription factor
nuclear factor-kB. Mol. Pharmacol. 52: 464–472
Lowenstein, C. J., Hill, S. L., Lafond-Walker, A., Wu, J., Allen, G.,
Landavere, M., Rose, N. R., Herskowitz, A. (1996) Nitric oxide
inhibits viral replication in murine myocarditis. J. Clin. Invest.
97: 1837–1843
Marletta, M. A. (1993) Nitric oxide synthase structure and
mechanism. J. Biol. Chem. 268: 12231–12234
Marletta,M.A.,Yoon,P.S.,Iyengar,R.,Leaf,C.D.,Wishnok,J.S.
(1988) Macrophage oxidation of
L-arginine to nitrite and nitrate:
nitric oxide is an intermediate. Biochemistry 27: 8708–8711
Matev, M., Angelova, I., Koichev, A., Leseva, M., Stefanov, G.
(1982) Clinical trial of a Plantago major preparation in the
treatment of chronic bronchitis. Vitr. Boles. 21: 133–137
Moncada, S., Palmer, R. M., Higgs, E. A. (1991) Nitric oxide:
physiology, pathophysiology, and pharmacology. Pharmacol.
Rev. 43: 109–142
Murai, M., Tamayama, Y., Nishibe, S. (1995) Phenylethanoids
in the herb of Plantago lanceolata and inhibitory effect on
arachidonic acid-induced mouse ear edema. Planta Med. 61:
479–480
Nathan, C. (1992) Nitric oxide as a secretory product of mam-
malian cells. FASEB J. 6: 3051–3064
Nathan, C. J. (1997) Inducible nitric oxide synthase: what differ-
ence does it make? J. Clin. Invest. 100: 2417–2423
Ogawa, O., Umegaki, H., Sumi, D., Hayashi, T., Nakamura, A.,
Thakur, N. K., Yoshimura, J., Endo, H., Iguchi, A. (2000)
Inhibition of inducible nitric oxide synthase gene expression by
indomethacin or ibuprofen in -amyloid protein-stimulated
J774 cells. Eur. J. Pharmacol. 408: 137–141
Palmer, R. M., Ashton, D. S., Moncada, S. (1988) Vascular
endothelial cells synthesize nitric oxide from
L-arginine.
Nature 333: 664–666
Peris, J., Stu
¨
bing, G., Vanaclocha, B. (1995) In: Peris, J., Stu
¨
bing,
G., Vanaclocha, B. (eds) Fitoterapia aplicada. M.I.C.O.F.,
Valencia, Spain, p. 416
Rice-Evans, C. A., Miller, N. J., Paganga, G. (1996) Structure-
antioxidant activity relationships of flavonoids and phenolic
acids. Free Radic. Biol. Med. 20: 933–956
Ringbom, T., Segura, L., Noreen, Y., Perera, P., Bohlin, L.
(1998) Ursolic acid from Plantago major, a selective inhibitor
of cyclooxygenase-2 catalyzed prostaglandin biosynthesis.
J. Nat. Prod. 61: 1212–1215
Smith, W. L., Marnett, L. J., DeWitt, D. L. (1991)
Prostaglandin and thromboxane biosynthesis. Pharmacol.
Ther. 49: 153–179
Smith, W. L., Garavito, R. M., DeWitt, D. L. (1996)
Prostaglandin endoperoxide H synthases (cyclooxygenases)-1
and -2. J. Biol. Chem. 271: 33157–33160
390 E. Vigo et al
Tezuka, Y., Irikawa, S., Keneko, T., Banskota, A., Nagaoka, T.,
Xiong, Q., Hase, K., Kadota, S. (2001) Screening of Chinese
herbal drug extracts for inhibitory activity on nitric oxide
production and identification of an active compound of
Zanthoxylum bungeanum. J. Ethnopharmacol. 77: 209–217
Vigo, E., Cepeda, A., Gualillo, O., Perez-Fernandez, R. (2004)
In-vitro anti-inflammatory effect of Eucalyptus globulus and
Thymus vulgaris: nitric oxide inhibition in J774A.1 murine
macrophages. J. Pharm. Pharmacol. 56: 257–263
Virgili, F., Kobuchi, H., Packer, L. (1998) Procyanidins
extracted from Pinus maritime (Pycnogenol): scavengers of
free radical species and modulators of nitrogen monoxide
metabolism in activated murine RAW 264.7 macrophages.
Free Radic. Biol. Med. 24: 1120–1129
Wallace, J. L., Bak, A., McKnight, W., Asfaha, S., Sharkey, K. A.,
MacNaughton, W. K. (1998) Cyclooxygenase 1 contributes to
inflammatory responses in rats and mice: implications for
gastrointestinal toxicity. Gastroenterology 115: 101–109
Wang, P. F., Kang, J. H., Zheng, R. L., Yang, Z. H., Lu, J. F.,
Gao, J. J., Jia, Z. J. (1996) Scavenging effects of phenylpropa-
noid glycosides from Pedicularis on superoxide anion and
hydroxyl radical by the spin trapping method (95) 02255-4.
Biochem. Pharmacol. 51: 687–691
Wheeler, A. P., Bernard, G. R. (1999) Treating patients with
severe sepsis. N. Engl. J. Med. 340: 207–214
Xiong, Q., Kadota, S., Tani, T., Namba, T. (1996) Antioxidative
effects of phenylethanoids from Cistanche deserticola . Biol.
Pharm. Bull. 19: 1580–1585
Xiong, Q., Tezuka, Y., Kaneko, T., Li, H., Tran, L., Hase, K.,
Namba, T., Kadota, S. (2000) Inhibition of nitric oxide by
phenylethanoids in activated macrophages. Eur. J. Pharmacol.
400: 137–144
Anti-inflammatory effect of Pinus sylvestris and Plantago lanceolata 391
